Abstract:

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Abstract:

The electrophysiological mechanism of the sinus node automaticity was previously considered exclusively regulated by the so-called "funny current". However, parallel investigations increasingly emphasized the importance of the Ca-homeostasis and Na/Ca exchanger (NCX). Recently, increasing experimental evidence, as well as insight through mechanistic modeling demonstrates the crucial role of the exchanger in sinus node pacemaking. NCX had a key role in the exciting story of discovery of sinus node pacemaking mechanisms, which recently settled with a consensus on the coupled-clock mechanism after decades of debate. This review focuses on the role of the Na/Ca exchanger from the early results and concepts to recent advances and attempts to give a balanced summary of the characteristics of the local, spontaneous, and rhythmic Ca releases, the molecular control of the NCX and its role in the fight-or-flight response. Transgenic animal models and pharmacological manipulation of intracellular Ca concentration and/or NCX demonstrate the pivotal function of the exchanger in sinus node automaticity. We also highlight where specific hypotheses regarding NCX function have been derived from computational modeling and require experimental validation. Nonselectivity of NCX inhibitors and the complex interplay of processes involved in Ca handling render the design and interpretation of these experiments challenging.

Abstract:

Background and Purpose: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca handling and sarcolemmal ion channels. An important player of this crosstalk is the Na/Ca exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the I-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). Experimental Approach: Currents were measured by patch-clamp, Ca-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv . SAN model. Key Results: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (I) was previously inhibited and , the effect of I was augmented when the Ca handling was suppressed. Conclusion and Implications: We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between I and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of I or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Abstract:

Each heartbeat is initiated by cyclic spontaneous depolarization of cardiomyocytes in the sinus node forming the primary natural pacemaker. In patients with end-stage renal disease undergoing hemodialysis, it was recently shown that the heart rate drops to very low values before they suffer from sudden cardiac death with an unexplained high incidence. We hypothesize that the electrolyte changes commonly occurring in these patients affect sinus node beating rate and could be responsible for severe bradycardia. To test this hypothesis, we extended the Fabbri et al. computational model of human sinus node cells to account for the dynamic intracellular balance of ion concentrations. Using this model, we systematically tested the effect of altered extracellular potassium, calcium, and sodium concentrations. Although sodium changes had negligible (0.15 bpm/mM) and potassium changes mild effects (8 bpm/mM), calcium changes markedly affected the beating rate (46 bpm/mM ionized calcium without autonomic control). This pronounced bradycardic effect of hypocalcemia was mediated primarily by I attenuation due to reduced driving force, particularly during late depolarization. This, in turn, caused secondary reduction of calcium concentration in the intracellular compartments and subsequent attenuation of inward I and reduction of intracellular sodium. Our in silico findings are complemented and substantiated by an empirical database study comprising 22,501 pairs of blood samples and in vivo heart rate measurements in hemodialysis patients and healthy individuals. A reduction of extracellular calcium was correlated with a decrease of heartrate by 9.9 bpm/mM total serum calcium (p < 0.001) with intact autonomic control in the cross-sectional population. In conclusion, we present mechanistic in silico and empirical in vivo data supporting the so far neglected but experimentally testable and potentially important mechanism of hypocalcemia-induced bradycardia and asystole, potentially responsible for the highly increased and so far unexplained risk of sudden cardiac death in the hemodialysis patient population.

Abstract:

Microscope-integrated optical coherence tomography (OCT) can provide valuable feed- back about the surgical procedure during ophthalmic surgeries through the visualization of two-dimensional cross-sections. The emerging swept-source technology also enables the acquisition of real-time volume scans during surgery. However, these volumes are inherently distorted, which limits their applicability for assistance functions due to inaccurate distance measurements. The three main topics addressed in this master thesis are the distortion correc- tion of OCT volume scans, the parametrization of the refractive surfaces and the examination of the influence of the refractive indices on the reconstruction accuracy.In this work, the 3D distortion correction of volume scans is realized with stepwise ray tracing and surface fitting. This allows the correction of fan distortion resulting from the scan ray geometry, refraction at interfaces and optical path length (OPL) compensation. It is shown that the implemented methods can be applied to segmented OCT volume scans and simulated data too. For the compensation of the fan distortion, a two-axis geometrical calibration method is proposed. With this calibration, the reconstruction error of a planar test target can be decreased by one order of magnitude.The surface fitting and reconstruction accuracy of different surface parametrization methods are evaluated, with a focus on the comparison of Zernike and B-spline surface parametrization. Two different cornea geometries are investigated in the simulations, a conical one and a deformed one with regard to the intraoperative environment. In both cases, increasing the number of polynomial terms of the Zernike and B-spline fitting results in a more accurate reconstruction. For the simulations, where noise and other measurement errors are excluded, the RMS reconstruction error of the points of the pupil plane can be reduced to below 10 μm. The number of polynomials required for this accuracy is similar for the Zernike polynomials and the B-splines. Analysis of experiments with ex vivo porcine eyes show the same relationship between the complexity of the parametrization and reconstruction accuracy. No previous publications were found that compare Zernike and B-spline fitting of corneal surfaces.The connection between the estimation error of the refractive indices and the reconstruction error of the pupil plane is evaluated through simulation. In these simulations the refractive index of the aqueous humour has a four times higher impact on the reconstruction accuracy of the pupil plane than the refractive index of the cornea.